Thermodynamic Analysis of Protein-Nanoparticle Interactions Links Binding Affinity and Structural Stability

When nanoparticles and nanoplastics enter biological fluids, their surfaces are rapidly coated with proteins, forming a corona that governs biological responses. However, understanding protein-surface interaction energetics remains a significant challenge. Here, we examine how protein charge distribution affects adsorption to polystyrene nanoparticles (PSNPs) by generating a series of lysine-to-alanine variants of the GB3 protein. This approach is unique because it explores how systematic perturbations in a controlled model protein influence protein-surface interactions. Using isothermal titration calorimetry (ITC), we found that the K19A variant binds most strongly to both nonfunctionalized and carboxylate-functionalized PSNPs. ITC thermograms indicate that K19A forms a stable monolayer, while other variants exhibit multilayer adsorption. The folded protein structure suggests that removing lysine at position 19 creates a flatter, more neutral interaction surface that promotes efficient initial binding. Fluorescence denaturation experiments show that PSNPs destabilize GB3 protein variants, and the binding free energy correlates strongly with protein unfolding (<i>r</i> = 0.82, <i>p</i> < 0.01 for carboxylate-functionalized PSNPs and <i>r</i> = 0.76, <i>p</i> < 0.03 for nonfunctionalized PSNPs). These results reveal how protein stability and charge distribution shape adsorption thermodynamics, informing frameworks for predicting protein-surface interactions.